Properties of hydrodynamic J-type countercurrent chromatography for protein separation using aqueous two-phase systems: With special reference to constructing conical columns

- The conical column is constructed on J-type countercurrent chromatography (CCC).
- The CCC conical column shows decent stationary phase retention.
- The CCC conical column shows sound phase mixing.
- Experimental conical columns were constructed based on prior theoretical analysis.
- Proteins are successfully separated using constructed real conical columns.

Protein separation using hydrodynamic countercurrent chromatography (CCC), where low backpressure is inherent, is more challenging, more time consuming and more costly when compared with separating small molecules. The most hopeful approach is to rationally design suitable columns for already commercialized J-type CCC machinery. By comparing 3 column geometries (3D helix, 2D spiral and 3D cone), we firstly constructed the mechanical model tailored to the conical column on J-type CCC using aqueous two-phase system (ATPS) on protein separation. Aimed at mechanistically understanding hydrodynamic CCC, we then developed a semi-quantitative model to account for contributions of both hydrodynamic and hydrostatic forces to stationary phase retention, and have subsequently compared the modelling outcomes with experimental results. We practiced a methodology to delineate both phase mixing and stationary phase retention before committing to physically constructing CCC columns. Following theoretical analyses, we finally constructed conical columns for J-type CCC. Using model proteins (myoglobin and lysozyme) and with 2 ATPSs containing PEG1000 and phosphate, sound protein separation has been achieved (resolution reaches 1.5-2.0 and stationary phase retention also exceeds 40%) for the selected ATPSs and under a varied level of sample volumes and loadings.